T cell proliferation and the calculation of a stimulation index are one of the mostly widely used methods in both mouse and man to demonstrate antigen specific immunity. We propose to define a "second generation" assay to replace traditional lymphocyte proliferation assays using [3H]thymidine uptake. Multiparameter flow cytometry will be used to assess antigen-specific T cell proliferation by measuring incorporation of BrdU, a thymidine analog. In addition to proliferating T cells, surface markers associated with proliferating cells will be examined. Surface markers to be examined will include CO27, CD45RO/RA, and CO95. These are all markers that can be used, singly or in combination, to distinguish naive from memory cells. Antigen-specific BrdU incorporation will be used to identify proliferating antigen-responsive cells, so that analysis of other markers can be perfoffi1ed by gating on these cells. In a preliminary study using SEB as a stimulus, positive correlation was observed between flow cytometric measurement of proliferating CD4+ T cells by BrdU incorporation and a standard proliferation assay measuring [3H]thymidine uptake by PBMC. We propose to extend the use of BrdU proliferation assay using flow cytometry in assessing T cell proliferative responses to CMV, flu, and tumor antigens. The aim of this pilot project will be to correlate lymphocyte proliferation assay using non-radioactive BrdU incorporation measured by multiparametric flow cytometry and a standard radioactive [3H]thymidine incorporation assay in the following systems: CMV, influenza, and at least one of the candidate cancer antigens to be evaluated by the consortium (MAGE-3, HER2, CEA, or gp100).